A helper T cell (Th cell) is known as a cell which regulates immune reactions in a body. Via an interaction resulting from the contact between cells or via a release of a substance called cytokine, the helper T cell acts on and regulates a cell which executes an immune reaction.
A helper T cell can be classified, based on the type of the cytokine it produces, broadly into one of the two types, i.e., Th1 and Th2. This concept was proposed in mice for the first time in 1986 by Mosmann et al. They showed that, in a mouse long term-subcultured cell CD4 positive T cell line, a classification is possible into a type I helper T (Th1) cell which produces cytokines such as IL-2, a tumor necrosis factor TNF-β and interferon (IFN)-γ and a type II helper T (Th2) cell which produces cytokines such as IL-4, IL-5, IL-6 and IL-10. This theory was considered to be applicable only to the mouse long term subcultured cell line, and to be difficult to be applied to an in vivo cell. Nevertheless, a subsequent accumulation of findings revealed that the theory can be also applicable to an in-vivo helper T cell.
A further subsequent study revealed that, in many of in-vivo helper T cells, there are populations which can not be classified into any of the subsets proposed by Mosmann. This tendency is remarkable especially in humans rather than in mice, and it was revealed that there are many cells producing cytokines which overlap the both subsets. Then a helper T cell subgroup exhibiting a combination of both patterns was designated as a Th0 cell.
The Th0 cell is considered to be a precursor before differentiation into Th1/Th2. This Th cell subgroup is not homogeneous, and involves a quantitative or qualitative difference in the cytokines produced by each Th0 cell. Accordingly, on the basis of the polarization between the Th1 cell and the Th2 cell, the functions of a Th cell in humans is not considered to be as simple as that in the mouse system. Although the findings in the immune system obtained in the animal experiments such as those in mice have been accumulated, they should carefully be applied to the pathological state of a disease in humans, and a study focusing on a human immune system is required.
A Th1 cell is considered to induce a cellular immunity via the activity of the cytokines it produces (IL-2, TNF-β, IFN-γ). In this context, the cellular immunity is a concept which means an immune reaction undertaken mainly by cells such as cytotoxic T cell (CTL), natural killer (NK) cell and macrophage, which is an immune reaction characterized by an action exerted by these cells directly toward a target. Examples are a phagocytic effect of a macrophage on an invading microorganism, an apoptosis-induced removal of a virus-infected cell by a cytotoxic T cell, a cytotoxic effect of an NK cell on a virus-infected cell, tumor cell or implanted myelocyte. This immune reaction is considered to be involved greatly in an immune reaction toward an intracellular parasite, virus or tumor, in an immune reaction toward a implanted graft, and in an organ-specific autoimmune disease.
IFN-γ which is a cytokine produced by a Th1 cell promotes the differentiation, proliferation, and activation of a cell executing a cellular immunity, whereby directing an in-vivo immune reaction toward the cellular immunity (hereinafter referred to as “type Th1 immune reaction”). A cytokine produced by a Th2 cell described below is known to inhibit a type Th1 immune reaction.
On the contrary to a Th1 cell, a Th2 cell is considered to induce an immune reaction such as a humoral immunity and allergic reaction via the activity of the cytokines it produces (IL-4, IL-5, IL-6, UL-10 and IL-13). The cells involved in these immune reactions are mainly B cells, eosinophiles and mast cells.
In this context, the humoral immunity is a concept which means an immune reaction capable of being transferred to another individual by injecting a serum, which is an immune reaction undertaken mainly by an antibody, i.e., IgG or IgM. The IgG and IgM are involved in a neutralization of a toxin by a cell or in microorganism aggregation or opsonin derivatization, the elimination of which by a macrophage is promoted by its activity.
On the other hand, the secretion of IgE, an antibody which mediates a type I allergic reaction, is promoted by the action of a Th2. The IgE binds to an IgE receptor (Fcε RI) on the surface of a mast cell, and employs as a trigger the crosslinking of an antigen-specific Fcε RI to allow various mediators (histamine, protease, heparin and the like) to be released from the mast cell, whereby inducing an antigen type I allergic reaction.
A cytokine produced by a Th2 cell promotes the humoral immunity or an allergic reaction-involved cell differentiation, proliferation, and activation, whereby directing an in-vivo immune reaction toward such an “antibody-mediated” immune reaction (hereinafter referred to as “type TH2 immune reaction”). A Th1 cell is known to inhibit a type Th2 immune reaction via the cytokines it produces (mainly IFN-γ).
The balance between the Th1 cell and the Th2 cell activation described above or the balance in the cell populations is considered to determine whether an in-vivo immune reaction is type Th1-dominant or type Th2-dominant. It is also recently thought that this balance determines the pathological state of a disease in the immune system.
Such findings are supported by an experiment in which the difference in the genetic background between various experimental mice resulted in a species-related deviation of the immune reaction to type Th1 or Th2 even when the infection origin was identical and the prognosis of the infection also varied.
For example, a C57BL/6 mouse exhibiting a type Th1 immune response to leishmaniasis, which is a type of parasitic infectious diseases, was resistant to the infection, while a Balb/c mouse exhibiting a type Th2 immune response was susceptible to the infection (Heinzel, F. P., Sadick, M. D., Holandy, B. J., et al., Exp. Med. 169:59, 1989). This may be resulting from an advantageous action of the type Th1 immune reaction in preventing the infection in a case of an intracellular infection, which destroys and eliminates a parasite together with a parasitized cell itself.
In addition, the C57BL/6 mouse became susceptible to the infection when treated with IL-4, IL-10 and anti-IFN-γ neutralizing antibody to impart with a type Th2 immune response, while the Balb/c mouse became resistant when treated with IFN-γ, anti-IL-4 neutralizing antibody and anti-IL-10 neutralizing antibody to impart with a type TH1 immune response. Based on such findings, a pathological state was revealed to be affected greatly by the type of the immune response brought by the genetic background of each mouse. Also against an intracellular parasitic pathogen such as those for listeriosis or brucellosis, the prevention of the infection was reported to be effected in a type Th1 immune response (Araya L N, Elzer P H, Rowe G E, Enright F M, Winter A J; J. Immunol. 143:10, 3330–7, 1989).
In an actual immune system, it has been revealed that biophlaxis is performed by controlling the balance between Th1 and Th2 with time.
Extrapolating with these, a control of an in vivo Th1/Th2 balance by the administration of a cytokine or an anti-cytokine antibody is expected to be an effective therapy against an abnormality in the immune system.
While the Th1 and Th2 are grouped by the difference in the cytokines they produces, the difference in the molecule on the surface of each cell (surface marker) has been investigated. If such a difference can be identified, it can serve as a basis for separating each cell to allow an intensive analysis to be performed. Especially if a surface molecule which transmits the signals for differentiation, proliferation or activation specifically to the Th1 or Th2 can be identified, it can directly be a target of the therapy.
However, a surface marker allowing an in-vivo Th1 or Th2 to be distinguished readily has not been established, but there are an increasing number of the reports relating to the surface markers of the Th1 and Th2 in recent years. As a hopeful candidate of the surface marker, a protein molecule expressed specifically on the surface of the Th2 was identified and designated as ST2L (Yanagisawa, K., et al.: FEBS Lett. 318:83, 1993, and Yanagisawa, K., et al.: J. Biochem. 121: 95, 1997).
An ST2L is one of the ST2 gene expression products. The ST2 gene expression products are grouped into the three types discussed below, each of which is considered to be generated by a selective splicing. The first type of the ST2 gene products is a soluble secretion type designated as ST2. This gene was identified earliest among the three types. An ST2 is also referred to as T1, Fit-1 or DER4, and classified into a delayed-early serum reaction gene group in the cell proliferation. Thus, the ST2 is not expressed in a Balb/c-3T3 cell in the G0 phase, but is expressed when a cell is stimulated to proliferate by serum and enters in the phase during the cell cycle of the G1 or S phase subsequent to the G0 phase, and peaked 10 hours after addition of the serum. This protein was identified by us, and we analyzed a gene encoding a human ST2 (see JP-A-6-178687).
The second type is a transmembrane receptor type ST2L. The ST2L has an extremely high homology in the amino acid sequence with an Interleukin-1 (IL-1; a molecule known to be involved in an inflammatory reaction) receptor, with a higher homology being in the intracellular region. Another molecule having a high homology with the IL-1 receptor is an IL-18 receptor, which is a member of a IL-1 receptor family.
It is known that a high affinity receptor is formed by the IL-1 receptor together with a protein AcP or by the IL-18 receptor together with a protein AcPL. Thus, it is suggested that ST2L may form a high affinity receptor together with a protein analogous to the Acp and the like.
Studies on the binding of the ST2L and the IL-1 receptor ligand revealed no binding with any of IL-1α, β receptor antagonist, indicating no clear involvement of IL-1 in a signal transmission pathway.
Studies on a ligand specific to the ST2L include a report of a binding protein purification or cloning, but the cloned protein may just be a binding protein which can not induce any signal, suggesting that a physiological ligand may elsewhere be existed.
In addition, the third type of the ST2 is a variant form of the first type, and designated as ST2V.
We investigated the ST2 expression by performing an RT-PCR using a hematopoietic cultured cell in the previous study. As a result, the ST2 gene was expressed in almost all cells except for lymphocytes.
In lymphocytic series, no expression was found in a cell of the B cell line, while a cell expressing the ST2 gene was found in the T cell line.
For the purpose of a further investigation in the T cell line, we examined the cells for the expression of the ST2 and ST2L in the mouse T cell line using a northern blotting method. As a result, the Th1 cell exhibited no expression of the ST2 even after the simultaneous stimulation with a phorbol ester (PMA) and A23187, while the Th2 cell expressed an mRNA of the ST2L even before any stimulation. After the stimulation, the expression of an mRNA of the ST2 was further induced.
Moreover, we performed an experiment using an EL-4 which. is a mouse cultured cell capable of being differentiated into both of the types Th1 and Th2, and found that the EL-4 when stimulated only by PMA produced the type Th1 cytokines such as IL-2 and IFN-γ, while it produced the type Th2 cytokines such as IL-4 and IL-5 when stimulated simultaneously by PMA and dibutyryl cAMP. In this process, no ST2 expression was observed without stimulation, but the expression of the mRNAs of the both of the ST2 and ST2L was induced strongly only when performing a simultaneous stimulation with PMA and dibutyryl cAMP. Based on these findings, the expression of the ST2 exhibits the same behavior as the expression of a type Th2 cytokine in this experimental model.
Also other groups reported that in mice the ST2L protein was expressed constitutively in the Th2 cell unlike to the cytotoxic T cells, and that an analysis using a flow cytometry revealed that the ST2L protein was capable of utilizing as a marker for a Th2 subset (Yanagisawa, K., Naito, Y., Kuroiwa, K. et al., J. Biochem., 121:95, 1997).
Xu et al. found in their study on the ST2 that an antibody against the ST2 induced an in vivo cell death in a Th2 subgroup (Xu D., et al. J.Exp.Med., 787–794: 187, 1998). In this in vivo experiment using the antibody, this antibody was proven to inhibit the susceptibility to the infection with leishmaniasis in Balb/c mice. While the Balb/c strain is susceptible to the infection since it exhibits a type Th2 immune response in leishmaniasis as described above, the type Th1 immune response is considered to be induced by the removal of the Th2 cell by the anti-ST2 antibody. In addition, this antibody exacerbated a collagen-induced arthritis in DBA-1 mice which is considered to be induced in the condition where the type Th1 immune reaction is dominant. These findings indicate that this anti-ST2 antibody inhibited the Th2 whereby inducing the type Th1 immune reaction. Nevertheless, the physiological function of this antibody has not been clarified.
In addition, a reduction in the eosinophile count, in the tissue staining-based Th2 count and in the secretion of the type Th2 cytokines such as IL-4, IL-5, IL-6 and IL-13 in the bronchial washing fluid in a model mouse whose allergic airway inflammation had been induced by administering the anti-ST2 antibody or ST2 was reported (Lohning M. et al.: Proc. Natl. Acad. Sci. USA, 6930–6935: 95, 1998, Xu D. et al.: J. Exp. Med. 787–794: 187, 1998). Based on these reports, it was suggested that an ST2L-mediated signal was inhibited as a result of a competition with a physiological ligand to which the secretion type ST2 might naturally been bound.
The experiments discussed above suggest that the ST2L is associated positively not only with Th1/Th2 balancing but also with Th2 functioning.
The secretion type ST2 is considered to correspond to the extracellular region of the transmembrane type ST2L. As discussed above, both are considered to be generated by a selective splicing.
While it is known that in the Th2 cell the ST2L is expressed constantly but the expression of the ST2 depends on an antigen stimulation, the physiological functions of the ST2 is not known currently. However, as discussed above, since an administration of the ST2 or an administration of a neutralizing antibody against the ST2L inhibits an allergic disease, i.e., a type Th2 immune reaction in a model mouse, a model may be possible in which the secretion type ST2 and the transmembrane type ST2L in blood compete with each other in binding to the physiological ligand and the signal for inducing a type Th2 immune reaction which is transmitted by the ST2L to the inside of a cell is inhibited by the ST2.
Thus, the secretion type ST2 is suggested to be involved in the signal transmission in an immune system and the in-vivo ST2 amount is suggested to serve as a novel index which reflects the condition of the immune system. Nevertheless, no satisfactory biological data have been obtained currently with regard to the secretion type ST2. Moreover, most of the studies on the Th1/Th2 have been conducted in experimental animals such as a mouse, and the resultant data should be applied to humans only after elucidating the functions specific to a human ST2.
For the purpose of accumulating the biological data of the human ST2 and elucidating the in-vivo functions, it is desired to develop a method for measuring the human ST2 in an in-vivo condition, i.e., in a non-denatured state, rapidly and conveniently. As such a method, immunological methods such as an ELISA utilizing a monoclonal antibody against the human ST2 are considered to be effective.
The monoclonal antibody against the human ST2 was attempted to be produced by us, and several types of the anti-human ST2 monoclonal antibodies have been obtained (Yoshida K. et al.,: Hybridoma 419–427:14, 1995). However, such antibodies were obtained using as an immunogen a human ST2 protein obtained by expressing a human ST2 gene in E.coli, and bind specifically to a denatured human ST2 but could not recognize a non-denatured human ST2 specifically. Accordingly, they could not be employed for example in an ELISA for measuring a non-denatured human ST2 in a biological sample.
On the other hand, Werenskiold et al. disclosed a method for measuring a human ST2 by means of a PCR method for detecting an mRNA or by means of an immunohistological staining (WO98/430990), but such a method was only qualitative or semi-quantitative, and was not intended to measure the human ST2 quantitatively.